RESUMO
Although estrogens exert a pronounced protective effect on multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), their therapeutic application has been limited by undesirable side effects thought to be mediated primarily through estradiol binding to intracellular estrogen receptor alpha. In this study, we found that signaling through the putative membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), was sufficient to mediate protection against EAE, which was significantly impaired in GPR30 gene-deficient mice. Treatment with G-1, an agonist that selectively activates GPR30 without engagement of the intracellular estrogen receptors, retained the ability of estradiol to protect against clinical and histological EAE without estradiol-associated side effects, deviated cytokine profiles, and enhanced suppressive activity of CD4(+)Foxp3(+) T regulatory cells through a GPR30- and programmed death 1-dependent mechanism. This study is the first to evaluate the protective effect of GPR30 activation on EAE, and provides a strong foundation for the clinical application of GPR30 agonists such as G-1 in multiple sclerosis.
Assuntos
Antígenos de Superfície/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/prevenção & controle , Proteínas de Membrana/fisiologia , Receptores de Estrogênio/fisiologia , Receptores Acoplados a Proteínas G/fisiologia , Regulação para Cima/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Superfície/fisiologia , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/fisiologia , Células Cultivadas , Ciclopentanos/farmacologia , Encefalomielite Autoimune Experimental/metabolismo , Estradiol/fisiologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Dados de Sequência Molecular , Receptor de Morte Celular Programada 1 , Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/deficiência , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Regulação para Cima/genéticaRESUMO
The mechanism by which oestrogens suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is only partially understood. We here demonstrate that treatment with 17beta-oestradiol (E(2)) in C57BL/6 mice boosted the expression of programmed death 1 (PD-1), a negative regulator of immune responses, in the CD4(+) FoxP3(+) regulatory T (Treg) cell compartment in a dose-dependent manner that correlated with the efficiency of EAE protection. Administration of E(2) at pregnancy levels but not lower concentrations also enhanced the frequency of Treg cells. Additionally, E(2) treatment drastically reduced the production of interleukin-17 (IL-17) in the periphery of immunized mice. However, E(2) treatment did not protect against EAE or suppress IL-17 production in PD-1 gene-deficient mice. Finally, E(2) failed to prevent Treg-deficient mice from developing spontaneous EAE. Taken together, our results suggest that E(2)-induced protection against EAE is mediated by upregulation of PD-1 expression within the Treg-cell compartment.
Assuntos
Antígenos de Superfície/biossíntese , Proteínas Reguladoras de Apoptose/biossíntese , Encefalomielite Autoimune Experimental/prevenção & controle , Estradiol/uso terapêutico , Interleucina-17/biossíntese , Animais , Antígenos de Superfície/genética , Proteínas Reguladoras de Apoptose/genética , Células Cultivadas , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos/métodos , Encefalomielite Autoimune Experimental/imunologia , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Morte Celular Programada 1 , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Regulação para Cima/efeitos dos fármacosRESUMO
Recombinant T-cell receptor ligands (RTLs) can prevent and reverse clinical and histological signs of experimental autoimmune encephalomyelitis (EAE) in an antigen-specific manner and are currently in clinical trials for treatment of subjects with multiple sclerosis (MS). To evaluate regulatory mechanisms, we designed and tested RTL551, containing the alpha1 and beta1 domains of the I-A(b) class II molecule covalently linked to the encephalitogenic MOG-35-55 peptide in C57BL/6 mice. Treatment of active or passive EAE with RTL551 after disease onset significantly reduced clinical signs and spinal cord lesions. Moreover, RTL551 treatment strongly and selectively reduced secretion of interleukin-17 and tumor necrosis factor alpha by transferred green fluorescent protein-positive (GFP+) MOG-35-55-reactive T-cells and almost completely abrogated existent GFP+ cellular infiltrates in affected spinal cord sections. Reduced inflammation in spinal cords of RTL551-treated mice was accompanied by a highly significant downregulation of chemokines and their receptors and inhibition of VCAM-1 (vascular cell adhesion molecule-1) and ICAM-1 (intercellular adhesion molecule-1) expression by endothelial cells. Thus, RTL therapy cannot only inhibit systemic production of encephalitogenic cytokines by the targeted myelin oligodendrocyte glycoprotein-reactive T-cells but also impedes downstream local recruitment and retention of inflammatory cells in the CNS. These findings indicate that targeted immunotherapy of antigen-specific T-cells can result in a reversal of CNS lesion formation and lend strong support to the application of the RTL approach for therapy in MS.
Assuntos
Movimento Celular/efeitos dos fármacos , Encefalomielite Autoimune Experimental/tratamento farmacológico , Fatores Imunológicos/farmacologia , Interleucina-17/metabolismo , Receptores de Antígenos de Linfócitos T/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Animais , Moléculas de Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/imunologia , Movimento Celular/imunologia , Quimiocinas/efeitos dos fármacos , Quimiocinas/imunologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/imunologia , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/fisiopatologia , Proteínas de Fluorescência Verde , Fatores Imunológicos/uso terapêutico , Imunoterapia/métodos , Interleucina-17/imunologia , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/imunologia , Esclerose Múltipla/fisiopatologia , Fragmentos de Peptídeos/farmacologia , Fragmentos de Peptídeos/uso terapêutico , Receptores de Antígenos de Linfócitos T/imunologia , Proteínas Recombinantes de Fusão/uso terapêutico , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Resultado do Tratamento , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Stroke induction in immunologically competent mice not only produces local ischemia and brain damage, but also induces early inflammatory changes in brain and peripheral immune responses. Although immune elements clearly are activated after brain vascular occlusion, the relative contribution of T and B lymphocytes to the developing lesion has not been quantified. We evaluated effects 22 h after middle cerebral artery occlusion (90 mins) on histologic injury and peripheral immune activation in severe combined immunodeficient (SCID) mice lacking T and B cells. Cortical and total infarct volumes were strikingly reduced in male SCID mice (n=14, 33+/-4% of contralateral cortex, n=10, 52+/-3% of contralateral hemisphere) versus immunologically intact C57BL/6 mice (wild type, n=9, 57+/-5% of contralateral cortex, 57+/-4% of contralateral hemisphere) (P<0.01). Striatal infarction was not altered (77+/-7% of contralateral striatum in SCID, 84+/-7% in wild type), suggesting that the core of the evolving ischemic lesion was not impacted by lack of T and B cells. As expected, inflammatory factors from immune cells in ischemic SCID brains were essentially absent, with the exception of interleukin-1beta increase in both SCID and wild type tissue. Spleen cell numbers were low in SCID mice, but were further reduced 22 h after stroke, with substantial reduction in most inflammatory factors except for increased expression of interferon-gamma and macrophage inflammatory protein (MIP)-2. These data quantify the damaging effect of T and B lymphocytes on early, evolving ischemic brain injury, and further implicate interleukin-1beta in brain and interferon-gamma and MIP-2 in spleen as inflammatory factors produced by cells other than T and B cells.
Assuntos
Linfócitos B/imunologia , Linfócitos B/fisiologia , Inflamação/imunologia , Inflamação/patologia , Acidente Vascular Cerebral/imunologia , Acidente Vascular Cerebral/patologia , Linfócitos T/imunologia , Linfócitos T/fisiologia , Animais , Encéfalo/patologia , Química Encefálica/fisiologia , Isquemia Encefálica/patologia , Contagem de Células , Infarto Cerebral/patologia , Quimiocinas/metabolismo , Citocinas/metabolismo , Citometria de Fluxo , Infarto da Artéria Cerebral Média/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , RNA/biossíntese , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Baço/citologia , Baço/imunologiaRESUMO
Females tend to have stronger Th1-mediated immune responses and are more prone to develop autoimmune diseases, including multiple sclerosis. Macrophages are major effector cells capable of mediating or modulating immune responses in experimental autoimmune encephalomyelitis (EAE). IL-13 and estrogen have opposing roles on macrophages (the former enhancing and the latter inhibiting) in terms of MHC class II (MHC II) up-regulation and, thus, these factors might influence susceptibility to EAE differently in females vs males. In accordance with this hypothesis, females lacking IL-13 displayed lower incidence and milder EAE disease severity than males after immunization with myelin oligodendrocyte glycoprotein (MOG)-35-55 peptide/CFA/pertussis toxin. Female IL-13 knockout (KO) mice with EAE consistently had reduced infiltration of CD11b(+) macrophages in the CNS along with significantly reduced expression of MHC II on these cells. Impaired MHC II expression was further corroborated upon LPS stimulation of female but not male bone marrow-derived CD11b(+) macrophages from IL-13KO mice, with restored expression after IL-13 pretreatment of female but not male macrophages. APCs from IL-13KO females induced less proliferation by MOG-35-55-reactive T cells, and splenocytes from MOG peptide-immunized females had lower expression of IL-12, IFN-gamma, MIP-2, and IFN-gamma-inducible protein 10 than males. In contrast, these splenocytes had higher expression of anti-inflammatory factors, IL-10, TGF-beta1, and FoxP3, a cytokine pattern typical of regulatory type II monocytes. These data suggest that the difference in EAE susceptibility in females is strongly influenced by gender-specific proinflammatory effects of IL-13, mediated in part through up-regulation of Th1-inducing cytokines and MHC II on CD11b(+) macrophages.
Assuntos
Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Predisposição Genética para Doença , Interleucina-13/fisiologia , Caracteres Sexuais , Animais , Apresentação de Antígeno/genética , Apresentação de Antígeno/imunologia , Células da Medula Óssea/imunologia , Células da Medula Óssea/metabolismo , Linhagem Celular , Células Cultivadas , Encefalomielite Autoimune Experimental/epidemiologia , Feminino , Incidência , Interleucina-13/deficiência , Interleucina-13/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
We previously demonstrated the therapeutic effects of MHC class II derived recombinant T cell receptor ligands (RTL), single-chain two domain complexes of the alpha1 and beta1 domains of MHC class II molecules genetically linked with an immunodominant peptide, in experimental autoimmune encephalomyelitis. In the current study, we produced a monomeric murine I-Aq-derived RTL construct covalently linked with bovine collagen type II peptide (bCII257-270) suitable for use in DBA/1LacJ mice that develop collagen-induced arthritis (CIA), an animal model of human rheumatoid arthritis, after immunization with bCII protein in CFA. In this study, we demonstrate that the I-Aq-derived RTLs reduced the incidence of the disease, suppressed the clinical and histological signs of CIA and induced long-term modulation of T cells specific for arthritogenic Ags. Our results showed that the I-Aq/bCII257-270 molecule could systemically reduce proinflammatory IL-17 and IFN-gamma production and significantly increase anti-inflammatory IL-10, IL-13, and FoxP3 gene expression in splenocytes. Moreover, I-Aq/bCII257-270 molecule could also selectively inhibit IL-1beta, IL-6, and IL-23 expression in local joint tissue. This is the first report demonstrating effective prevention of joint inflammation and clinical signs of CIA with an I-Aq-derived RTL, thus supporting the possible clinical use of this approach for treating rheumatoid arthritis in humans.
Assuntos
Artrite Experimental/prevenção & controle , Receptores de Antígenos de Linfócitos T/agonistas , Proteínas Recombinantes de Fusão/uso terapêutico , Animais , Formação de Anticorpos , Antígenos/imunologia , Artrite Experimental/imunologia , Artrite Experimental/patologia , Bovinos , Citocinas/genética , Citocinas/metabolismo , Expressão Gênica , Fator 3-gama Nuclear de Hepatócito/genética , Fator 3-gama Nuclear de Hepatócito/metabolismo , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/uso terapêutico , Humanos , Articulações/patologia , Ligantes , Camundongos , Camundongos Endogâmicos DBA , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapêutico , Baço/imunologia , Líquido Sinovial/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Regulação para CimaRESUMO
Inflammation results in CNS damage in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. It is uncertain how much repair of injured myelin and axons can occur following highly selective anti-inflammatory therapy in EAE and MS. In this study, SJL/J mice with established EAE were treated successfully with an antigen-specific recombinant T cell receptor ligand (RTL), RTL401, a mouse I-A(s)/PLP-139-151 construct, after the peak of EAE. To define the mechanisms by which late application of RTL401 inhibits EAE, we evaluated mice at different time points to assess the levels of neuroinflammation and myelin and axon damage in their spinal cords. Our results showed that RTL401 administered after the peak of acute EAE induced a marked reduction in inflammation in the CNS, associated with a significant reduction of demyelination, axonal loss and ongoing damage. Electron microscopy showed that RTL-treated mice had reduced pathology compared with mice treated with vehicle and mice at the peak of disease, as demonstrated by a decrease in continued degeneration, increase in remyelinating axons and the presence of an increased number of small, presumably regenerative axonal sprouts. These findings indicate that RTL therapy targeting encephalitogenic T cells may promote CNS neuroregenerative processes.